RAP, a member of the Ras superfamily of small GTPases and its putative GTPase activating proteins and guanine nucleotide exchange factors in raw 264.7 macrophages

Raw 264.7 ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Fcγ Receptor ◽

Raw 264.7 Macrophages ◽

Gtpase Activating Proteins ◽

Ras Superfamily

The Fcγ receptor is a cell surface protein essential in the immune response that binds IgG-opsonized particles resulting in phagocytosis. Phagocytosis is a process used to remove pathogens and confine them in a vacuole that will enable their breakdown. The members of the Ras superfamily of small G proteins have been identified in samples where the activated Fcγ receptor complex was captured and analyzed using tandem mass spectrometry. The protein Rap. beloning to the Ras superfamily, guanosine triphosphatases (GTPase) activating proteins (GAPs), which promote the dissociation of GTP, and guanine nucleotide exchange factors (GEFs), that permits the exchange of GDP for GTP, were detected by SEQUEST in RAW 264.7 macrophages and futher analyzed using various methods. In this study, Raps, RasGAPs, and RapGEFs, were observed by tandem mass spectrometry and sequence correlation analysis. The selected isoforms were confirmed by Western blots, live cell confocal microscopy with fluorescent fusion constructs and antibody staining to verify the localization of Ras proetins, specifically Rap1, p120RasGAP and C3G, a RapGEF, to activated Fc reeceptor [sic].

Hormones Secretion and Rho GTPases in Neuroendocrine Tumors

Cancers ◽

10.3390/cancers12071859 ◽

2020 ◽

Vol 12 (7) ◽

pp. 1859

Author(s):

Laura Streit ◽

Laurent Brunaud ◽

Nicolas Vitale ◽

Stéphane Ory ◽

Stéphane Gasman

Keyword(s):

Neuroendocrine Tumors ◽

Rho Gtpases ◽

Rho Gtpase ◽

Secretory Activity ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Gtpase Activating Proteins

Neuroendocrine tumors (NETs) belong to a heterogeneous group of neoplasms arising from hormone secreting cells. These tumors are often associated with a dysfunction of their secretory activity. Neuroendocrine secretion occurs through calcium-regulated exocytosis, a process that is tightly controlled by Rho GTPases family members. In this review, we compiled the numerous mutations and modification of expression levels of Rho GTPases or their regulators (Rho guanine nucleotide-exchange factors and Rho GTPase-activating proteins) that have been identified in NETs. We discussed how they might regulate neuroendocrine secretion.

Stress-dependent inhibition of cell polarity through unbalancing the GEF/GAP regulation of Cdc42

10.1101/2021.08.10.455852 ◽

2021 ◽

Author(s):

Clàudia Salat-Canela ◽

Mercè Carmona ◽

Rebeca Martín-García ◽

Pilar Pérez ◽

José Ayté ◽

...

Keyword(s):

Cell Polarity ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Gtp Hydrolysis ◽

Gtpase Activating Proteins ◽

Dependent Inhibition ◽

Stress Dependent

Cdc42 rules cell polarity and growth in fission yeast. It is negatively and positively regulated by GTPase-activating proteins (GAPs) and by Guanine-nucleotide Exchange factors (GEFs), respectively. Active Cdc42-GTP localizes to the poles, where it associates with numerous proteins constituting the polarity module. However, little is known about its down-regulation. We describe here that oxidative stress causes Sty1 kinase-dependent Cdc42 inactivation at cell poles. Both the amount of active Cdc42 at poles and cell length inversely correlate with Sty1 activity, explaining the elongated morphology of Δsty1 cells. We have created stress-blinded cell poles by either eliminating two Cdc42 GAPs or through the constitutive tethering of a GEF to the cell tips, and biochemically demonstrate that Rga3 is a direct substrate of Sty1. We propose that stress-activated Sty1 promotes GTP hydrolysis and prevents GEF activity at the cell tips, thus leading to the inhibition of Cdc42 and polarized growth cessation.

Activation of Rac and Cdc42 Video Imaged by Fluorescent Resonance Energy Transfer-Based Single-Molecule Probes in the Membrane of Living Cells

Molecular and Cellular Biology ◽

10.1128/mcb.22.18.6582-6591.2002 ◽

2002 ◽

Vol 22 (18) ◽

pp. 6582-6591 ◽

Cited By ~ 394

Author(s):

Reina E. Itoh ◽

Kazuo Kurokawa ◽

Yusuke Ohba ◽

Hisayoshi Yoshizaki ◽

Naoki Mochizuki ◽

...

Keyword(s):

Single Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Leading Edge ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Gtpase Activating Proteins

ABSTRACT Rho family G proteins, including Rac and Cdc42, regulate a variety of cellular functions such as morphology, motility, and gene expression. We developed fluorescent resonance energy transfer-based probes which monitored the local balance between the activities of guanine nucleotide exchange factors and GTPase-activating proteins for Rac1 and Cdc42 at the membrane. These probes, named Raichu-Rac and Raichu-Cdc42, consisted of a Cdc42- and Rac-binding domain of Pak, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras. With these probes, we video imaged the Rac and Cdc42 activities. In motile HT1080 cells, activities of both Rac and Cdc42 gradually increased toward the leading edge and decreased rapidly when cells changed direction. Under a higher magnification, we observed that Rac activity was highest immediately behind the leading edge, whereas Cdc42 activity was most prominent at the tip of the leading edge. Raichu-Rac and Raichu-Cdc42 were also applied to a rapid and simple assay for the analysis of putative guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) in living cells. Among six putative GEFs and GAPs, we identified KIAA0362/DBS as a GEF for Rac and Cdc42, KIAA1256 as a GEF for Cdc42, KIAA0053 as a GAP for Rac and Cdc42, and KIAA1204 as a GAP for Cdc42. In conclusion, use of these single-molecule probes to determine Rac and Cdc42 activity will accelerate the analysis of the spatiotemporal regulation of Rac and Cdc42 in a living cell.

Distinct Subclasses of Small GTPases Interact with Guanine Nucleotide Exchange Factors in a Similar Manner

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.7444 ◽

1998 ◽

Vol 18 (12) ◽

pp. 7444-7454 ◽

Cited By ~ 13

Author(s):

Gwo-Jen Day ◽

Raymond D. Mosteller ◽

Daniel Broek

Keyword(s):

Alpha Helix ◽

Small Gtpases ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Structural Determinants ◽

Ras Superfamily

ABSTRACT The Ras-related GTPases are small, 20- to 25-kDa proteins which cycle between an inactive GDP-bound form and an active GTP-bound state. The Ras superfamily includes the Ras, Rho, Ran, Arf, and Rab/YPT1 families, each of which controls distinct cellular functions. The crystal structures of Ras, Rac, Arf, and Ran reveal a nearly superimposible structure surrounding the GTP-binding pocket, and it is generally presumed that the Rab/YPT1 family shares this core structure. The Ras, Rac, Ran, Arf, and Rab/YPT1 families are activated by interaction with family-specific guanine nucleotide exchange factors (GEFs). The structural determinants of GTPases required for interaction with family-specific GEFs have begun to emerge. We sought to determine the sites on YPT1 which interact with GEFs. We found that mutations of YPT1 at position 42, 43, or 49 (effector loop; switch I), position 69, 71, 73, or 75 (switch II), and position 107, 109, or 115 (alpha-helix 3–loop 7 [α3-L7]) are intragenic suppressors of dominant interfering YPT1 mutant N22 (YPT1-N22), suggesting these mutations prevent YPT1-N22 from binding to and sequestering an endogenous GEF. Mutations at these positions prevent interaction with the DSS4 GEF in vitro. Mutations in the switch II and α3-L7 regions do not prevent downstream signaling in yeast when combined with a GTPase-defective (activating) mutation. Together, these results show that the YPT1 GTPase interacts with GEFs in a manner reminiscent of that for Ras and Arf in that these GTPases use divergent sequences corresponding to the switch I and II regions and α3-L7 of Ras to interact with family-specific GEFs. This finding suggests that GTPases of the Ras superfamily each may share common features of GEF-mediated guanine nucleotide exchange even though the GEFs for each of the Ras subfamilies appear evolutionarily unrelated.